1. Field of the Invention
This invention is related to a method for rapidly quantifying hydroxymethylated DNA by binding DNA to plastic carrier followed by immunodetection of 5-hydroxymethylcytosine or hydroxymethylcytosine structure that is a marker of DNA hydroxymethylation.
2. Description of the Related Art
DNA methylation is an epigenetic modification which is catalyzed by DNA cytosine-5-methyltransferases (DNMTs) and occurs at the 5-position (C5) of the cytosine ring, within CpG dinucleotides. DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation (Laird P W et al: Annu Rew. Genet, 1996; Reik W et al: Science, 2001; Robertson K D et al: Nature Rew. Genet, 2005). Alterations in DNA methylation have been demonstrated to cause change in gene expression. For example, hypermethylation leads to gene silencing or decreased gene expression while hypomethylation activates the genes or increases gene expression. Region-specific DNA methylation is mainly found in 5′-CpG-3′ dinucleotides within the promoters or in the first exon of genes, which is an important pathway for the repression of gene transcription in diseased cells.
Very recently, a novel modified nucleotide, 5-hydroxymethylcytosine (5-hmC) has been detected to be abundant in mouse brains and embryonic stem cells (Kriaucionis S et al: Science, 2009). 5-Hydroxymethylcytosine was first seen in bacteriophages in 1952 (Wyatt G R et al: Nature, 1952). In mammals, it can be generated by oxidation of 5-methylcytosine, a reaction mediated by the Tet family of enzymes and Dnmt proteins (Tahiliani M et al: Science, 2009). 5-hmC is a hydroxylated and methylated form of cytosine. The 5-hydroxymethylcytosine structure may include 5-methylhydroxycytidine, and 5-hydroxymethyl-2-deoxy-cytidine, and further include 5-hydroxymethyl-2-deoxy-cytidine monophosphate (hmdCMP), 5-hydroxymethyl-2-deoxy-cytidine diphosphate (hmdCDP), and 5-hydroxymethyl-2-deoxy-cytidine triphosphate (hmdCTP). The broader function of 5-hmC in epigenetics is still a mystery today. However, a line of evidence showed that 5-hmC plays a role in DNA demethylation, chromatin remodeling and gene expression regulation, specifically in brain-specific gene regulation (Valinluck V et al: Cancer Res, 2007, Valinluck V et al: Nucleic Acid res, 2004, Penn N W et al: Biochem J, 1976, Penn N W et al: biochem J, 1972):                1) Conversion of 5-methylcytosine (5-mC) to 5-hmC greatly reduced affinity of MBD proteins to methylated DNA;        2) The observation that formation of 5-hmC by oxidative damage or by the addition of aldehydes via Dnmts prevents Dnmt-mediated methylation of target cytosine.        3) 5-hmC may recruit specific binding proteins that alter chromatin structure or DNA methylation patterns.        4) 5-hmC accounts for roughly 40 percent of the methylated cytosine in Purkinje cells and 10 percent in granule neurons.        
Because of the presence of 5-hmC in DNA with an unclear function in gene regulation and the discovery of the enzymes that produce 5-hmC, it is considered important to know the distribution of this base in different cell types and in different compartments of the genome of mammalians. It is particularly important to identify hydroxymethylation status in human cell/tissues with and without diseases. Currently used methylated DNA analysis methods including restriction enzyme digestion, bisulfite or MeDIP-mediated MS-PCR and sequencing have been demonstrated to not be suitable for 5-hmC or hydroxymethylated DNA detection as 5-hmC and 5-mC are virtually indistinguishable with these methods (Huang Y et al: PLoS One, 2010; Jin S G et al: Nucleic Acid Res, 2010, Nestor C et al: BioTechniques, 2010). Several chromatography-based techniques such as HPLC and TLC mass spectrometry are used for detecting 5-hmC (Kriaucionis S et al: Science, 2009; Penn N W et al: biochem J, 1972). In the analysis, DNA is digested into single nucleotides and total genomic 5-hmC is quantified. However these methods are labor intensive, time-consuming, or require large amounts of DNA (>250 ng) as the starting material for measurement, or rely on the use of expensive equipment. These disadvantages lead to cost-ineffectiveness, low throughput and inconvenience for routine application in most health and medical institutions. Thus, there is a need to establish a method to improve the detection of 5-hmC or hydroxymethylated DNA.